DESCRIPTION: The central aim of this project is to explore in vivo the mechanisms underlying the formation of axonal terminal arborizations and the innervation of peripheral targets by central neurons. Specific leech central neurons (P neurons) extend axons early in development that pioneer the paths to the targets and are the first to establish terminal fields. In the process of generating these fields, homologous P cells inhibit each other from growing extensively in each other's territory. Later-growing neurons (AP cells) use these pioneers as substrates for growth to and branching at the target area; in fact, the pioneer is both necessary and sufficient for the generation of normal Ap cell arbors. These results indicate that a series of enhancing and/or inhibiting interactions modulate the extension, retraction and branching of neurites during the establishment of peripheral arbors by leech central neurons. The working hypotheses of this project are that these interactions (a) modulate neurite growth by affecting the assembly of cytoskeletal components, and (b) are mediated at least in part by changes in intracellular calcium levels. The experiments proposed in this grant are designed to test these hypotheses and to provide the bases for future examination of the mechanisms involved in the signaling and responses of these cells the principal experimental approaches will be (1) to study the kinematics of arbor growth using time-lapse analysis of dye-filled embryonic neurons in intact animals, (2) to determine the arrangement of cytoskeletal elements at various stages of growth and after experimental manipulations by injecting tagged actin and tubuli subunits into individual growing neurons, and by electron microscopy, and (3) to search for relations between changes in cytoskeletal arrangements and intracellular calcium concentration. These studies will provide further insight into production and selective retraction of axonal projections, a common and important feature of the innervation of peripheral tissues.